Entrapped Beta-Cyclodextrin Polymer and a Method of Preparing the Same

ABSTRACT

Provided are entrapped β-cyclodextrin polymers and a method of preparing the same. Particularly, entrapped β-cyclodextrin polymers are prepared by adding epichlorohydrin to β-cyclodextrin to prepare a polymer in a gel state, entrapping the polymer by entrapment of metal ions on the surface of the polymer and pulverizing the entrapped polymer to prepare powders. According to the entrapped β-cyclodextrin polymers, steroid hormones contained in a biological sample can be simply extracted without using an additional device required in conventional solid-phase extraction.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to, and the benefit of, Korean PatentApplication No. 10-2006-0119300, filed on Nov. 29, 2006, the entiredisclosure of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an entrapped β-cyclodextrin polymersand a method of preparing the same.

2. Description of the Related Art

In general, solid-phase extraction (SPE) via hydrophobic interactionsbetween a packing material and an analyte has been widely used toextract steroid hormones from biological fluids such as urine and bloodfor clinical trials, food analyses, environmental analyses, drug tests,and doping controls for athletes (Journal of Chromatography A, 885:3-16, 237-250, 321-341, 2000; and Rapid Communications in MassSpectrometry, 16: 2221-2228, 2002).

In the SPE of steroid hormones using hydrophobic interactions, amodified silica such as such as C8, C18, phenyl and amino and acopolymer such as XAD (styrene-divinylbenzene copolymer) and Oasis HLB™(divinylbenzene-N-vinylpypyrrolidone copolymer) are generally used as anadsorbent. However, the silica adsorbent has a drawback, that it isunstable at extreme pH condition during pre-treatment of the sample,becomes deactivated, and the copolymer adsorbent has a drawback that itsuse is limited in pre-treatment of compounds having high polarityalthough it is not affected by any pH conditions.

When steroid hormones extracted from biological fluids are analyzed,they are extracted by SPE in pre-treatment and separated from variousinterfering substances by gas or liquid chromatography combined withmass spectrometry (GC-MS or LC-MS). Reversed-phase liquid chromatography(RPLC), one of the most widely used techniques to separate steroidhormones from interfering backgrounds in the samples, also employshydrophobic interaction (Journal of Chromatography A, 935: 141-172,2001). Accordingly, biological substances having different chemicalproperties from the steroids are easily removed by SPE during thepre-treatment process. However, biological substances having similarchemical properties to the steroids are extracted with the steroids andhave similar retention time during separation in RPLC.

To overcome the problems, an immuno-affinity chromatography based onbiological specificity has been used (Journal of Chromatography A, 794:37-43, 1998; Rapid Communications in Mass Spectrometry, 16: 370-374,2002); however, it is limited because of time-consuming process toobtain antibodies of steroid hormones and the high cost of the process.

Meanwhile, β-cyclodextrin, unlike the conventional adsorbents, iscapable of sequestering an organic compound having a particularstructure or a steroid hormone within its inner cavity (Journal ofSeparation Science, 25: 789-813, 2002; Steroids, 68: 321-327, 2003).

Korean Patent Application Nos. 1997-0018599, 1997-0037127 and1997-0037128 disclose methods of removing cholesterol, a startingsubstance to make steroid hormones from a liquid such as milk and creamusing the property of β-cyclodextrin. However, the methods have adrawback that a small amount of β-cyclodextrin still remains even aftercentrifugation in the process of separating the β-cyclodextrin, to whichcholesterol is adsorbed, from the liquid such as milk and cream from theliquid.

To solve the above problem, there was introduced a method to removecholesterol having a structure similar to that of steroid hormones frommilk by chemically immobilizing β-cyclodextrin onto glass beads as a SPE(Archives of Pharmaceutical Research, 27: 873-877, 2004). However, thismethod is not efficient because the activity of β-cyclodextrin, which ischemically immobilized onto the glass beads, is reduced due to chemicaltreatments and thus cholesterol cannot be removed efficiently.

In addition, there was introduced a method to increase cholesterolremoval rate by forming cross-links among β-cyclodextrin molecules usingepichlorohydrin as a chemical linker in an aqueous NaOH solution toincrease β-cyclodextrin activity (Archives of Pharmaceutical Research,27: 1183-1187, 2004). Various polymerized β-cyclodextrins have beenprepared by applying various conditions during the cross-linking ofβ-cyclodextrin molecules and physical properties and activities of theβ-cyclodextrin have been measured (Polymer International, 54: 744-753,2005). However, the polymerized β-cyclodextrin prepared using theepichlorohydrin forms a gel state which becomes swollen when used inextracting steroid hormones. Thus, it gives a problem to use only a verysmall amount of the polymerized β-cyclodextrin in the pre-treatmentrelative to the amount of the sample.

SUMMARY OF THE INVENTION

As a result of intensive researches to solve the drawbacks of theconventional processes, the present inventors have found that a complexof β-cyclodextrin polymer and steroid hormones is formed in biologicalfluids without being swollen by adding a metal salt solution toβ-cyclodextrin polymer in a gel state prepared using epichlorohydrin toentrap β-cyclodextrin polymer on the surface of the β-cyclodextrinpolymer and powderizing the entrapped β-cyclodextrin polymer, and thussteroid molecules can be efficiently extracted.

The entrapped β-cyclodextrin polymers according to the present inventioncan be applied to prepare a packing material for solid-phase extraction.Steroid compounds can be simply separated from a complex ofβ-cyclodextrin by a simple method of forming the complex ofβ-cyclodextrin as well as steroid compounds by adding entrappedβ-cyclodextrin polymers prepared according to the present invention tobiological fluids containing various steroid compounds are extractedfrom the inclusion complex using an organic solvent.

The present invention relates to entrapped β-cyclodextrin polymers whichcan be used to prepare a packing material for solid-phase extractioncapable of easily extracting steroid compounds.

In addition, steroid compounds can be efficiently extracted frombiological samples according to the present invention. The presentinvention can be applied to clinical researches on various types ofendocrine-dependent diseases since steroid hormones including androgen,estrogen and corticoids can be selectively extracted from biologicalfluids such as urine.

In addition, a sample in which steroid compounds are removed can beprepared as desired since steroid compounds can be selectively extractedaccording to the present invention.

In addition, the present invention can be applied to doping control ofathletes since synthetic anabolic steroids can be effectively extracted.

Therefore, the present invention provides a method for preparation ofentrapped β-cyclodextrin polymers easily adsorbing and desorbing steroidcompounds.

According to an aspect of the present invention, there are providedentrapped β-cyclodextrin polymers, wherein metal ions are coated on thesurface of the entrapped β-cyclodextrin polymers formed by the reactionof β-cyclodextrin monomers and epichlorohydrin.

According to another aspect of the present invention, there is provideda method for preparation of entrapped β-cyclodextrin polymers, themethod comprising: preparing a polymer in a gel state by dissolvingβ-cyclodextrin in an aqueous alkali solution and adding epichlorohydrinto the solution; preparing entrapped β-cyclodextrin polymers byimmersing the β-cyclodextrin polymers in a gel state in a metal saltsolution followed by coating and entrapping on the surface of theβ-cyclodextrin polymers; and washing, drying and pulverizing theentrapped polymer to powderize the entrapped β-cyclodextrin polymers.

According to another aspect of the present invention, there is provideda packing material for solid-phase extraction comprising the entrappedβ-cyclodextrin polymers.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features and advantages of the present inventionwill become more apparent by describing in detail exemplary embodimentsthereof with reference to the attached drawings in which:

FIG. 1A shows a chemical structure of β-cyclodextrin and FIG. 1Bschematically shows a three-dimensional structure of β-cyclodextrin;

FIG. 2 shows a flow diagram for a method of preparing entrappedβ-cyclodextrin polymers according to Example 1;

FIG. 3A shows a scanning electron microscope (SEM) image ofβ-cyclodextrin, FIG. 3B shows a SEM image of entrapped β-cyclodextrinpolymers prepared in Example 1, and FIG. 3C indicates a result ofelemental analysis showing that Ca ions which is an entrapping agent isdistributed on the surface of the β-cyclodextrin polymers;

FIGS. 4A and 4B show extracted-ion chromatograms illustrating results ofchromatographic analyses of endogenous androgen and estrogen extractedfrom urine according to Example 2;

FIGS. 5A, 5B, 5C and 5D show extracted-ion chromatograms illustratingresults of chromatographic analyses of synthetic anabolic steroid indoping control, which is extracted from urine according to Example 3;and

FIG. 6 shows chromatograms illustrating results of chromatographicanalyses of urinary corticoids according to Example 4.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in detail withreference to the accompanying drawings, in which exemplary embodimentsof the invention are shown.

The present invention relates to entrapped β-cyclodextrin polymers,wherein metal ions are coated on the surface of the entrappedβ-cyclodextrin polymers formed by the reaction of β-cyclodextrinmonomers and epichlorohydrin.

The present invention relates to a method of preparing entrappedβ-cyclodextrin polymers, the method comprising:

preparing β-cyclodextrin polymers in a gel state by dissolvingβ-cyclodextrin in an aqueous alkali solution and adding epichlorohydrinto the solution;

preparing entrapped β-cyclodextrin polymers by immersing theβ-cyclodextrin polymers in a gel state in a metal salt solution followedby coating and entrapping on the surface of the β-cyclodextrin polymers;and

washing, drying and pulverizing the entrapped polymers to powderize theentrapped β-cyclodextrin polymers.

The present invention relates to a method for selective extraction ofsteroid compounds using entrapped β-cyclodextrin polymers. The entrappedβ-cyclodextrin polymers are prepared by adding epichlorohydrin toβ-cyclodextrin to prepare a polymer in a gel state, entrapping thepolymer with metal ions by coating them on the surface of the polymer,and pulverizing the product.

FIGS. 1A and 1B show β-cyclodextrins used in the present invention. FIG.1A shows a chemical structure of β-cyclodextrin and FIG. 1Bschematically shows a three-dimensional structure of P3-cyclodextrin.The β-cyclodextrin is α-1,4-linked cyclic oligosaccharide with sevenglucose units containing hydrophilic exterior and hydrophobic interiorcavity (FIG. 1A). Since a hydrophobic cavity is formed in theβ-cyclodextrin and hydrophilic functional groups are exposed on thesurface of the β-cyclodextrin, the β-cyclodextrin is responsible forformation of inclusion complexes with guest molecules throughnon-covalent interactions (FIG. B).

β-cyclodextrin becomes more widely used due to its particular structureand the property of forming an inclusion complex, but it has a problemof swelling in an aqueous solution as described above.

The present invention relates to the entrapped β-cyclodextrin polymerthat is not swelled during sample preparation of urinary steroids, amethod of preparing the entrapped β-cyclodextrin polymers, and varioususes thereof. In the entrapped β-cyclodextrin polymers, it is formed bythe reaction of β-cyclodextrin monomers and epichlorohydrin followed byentrapment of metal ions.

A method of preparing entrapped β-cyclodextrin polymers according to thepresent invention includes: preparing a polymer in a gel state bydissolving β-cyclodextrin in an aqueous alkali solution and addingepichlorohydrin to the solution; entrapping the polymer by immersing thepolymer in a metal solution; and washing, drying and pulverizing theentrapped polymer to prepare products.

First, cross-links are formed among β-cyclodextrin monomers usingepichlorohydrin in order to bind the β-cyclodextrin monomers. Herein,the β-cyclodextrin monomers are dissolved in an aqueous alkali solutionto be used.

The alkali aqueous solution may be prepared using an alkali metalhydroxide, such as NaOH and KOH. If they have the same concentration, itis preferred to us NaOH solution because it has higher reactivity thanKOH solution. Then, metal ions are introduced into the process of curingthe polymerized β-cyclodextrin in the gel state in which cross-links areformed. The metal ions may be Ca or Na and a salt form of the metal isintroduced into the process. The metal salt may be selected from thegroup consisting of CaCl₂, NaCl, Ca(NO₃)₂ and CaSO₄, and CaCl₂ is usedto achieve the fast immersion of polymer if the metal salts have thesame concentration.

The concentration of the metal salt solution may be in the range of 0.05to 1.0 M, preferably 0.2 to 0.3 M. When the concentration is within therange above, the metal ions can be efficiently interposed between thecross-linked β-cyclodextrin layers.

Metal ions can be interposed between the cross-linked β-cyclodextrinlayers using the halogenated metal salts. FIGS. 3B and 3C show the Caions interposed between the layers. When the metal ions are interposedbetween the layers, the swelling does not occur any more and theselective binding of β-cyclodextrin to steroid compounds can beenhanced.

The entrapped β-cyclodextrin polymers sufficiently washed with ethanoland water, dried at a temperature in the range of 50 to 70° C., andpulverized using a variety of methods to prepare the products. Here, theentrapped β-cyclodextrin polymers have a diameter in the range of 100 to500 μm, preferably about 200 μm.

Thus prepared products of the present invention easily form aβ-cyclodextrin-steroid compound complex since they can be selectivelybound to a compound having a steroid structure. In addition, inclusivesteroids are easily isolated with an organic solvent and evaporating theorganic solvent.

Since the entrapped β-cyclodextrin polymers can extract and isolatevarious compounds having the steroid structure from biological fluids,it can be used to a variety of fields in which the steroid compoundneeds to be selectively isolated or removed.

For example, the entrapped β-cyclodextrin polymers of the presentinvention can be used as a packing material of a cartridge forsolid-phase extraction (SPE). Here, the particle size of the powders isrequired to be uniform in order to maximize extraction efficiency.

A method for preparing entrapped β-cyclodextrin polymers according to anembodiment of the present invention is schematically shown in FIG. 2.

Meanwhile, a method for extraction of steroid compounds using theproducts according to the present invention will be described.

First, a liquid sample containing steroid compounds is diluted using aphosphate buffer solution and an enzyme is added to hydrolyze. Thesample may be a liquid containing steroid compounds, for example, urine,blood, a tissue extract and a cell culture medium, which can be dilutedusing water or a phosphate buffer solution to prepare a solution in astate similar to urine (Rapid Communications in Mass Spectrometry, 16:2221-2228, 2002), and extracted in the same manner used in urine.

Here, the amount of entrapped β-cyclodextrin polymers may be in therange of 0.2 to 0.5 g per 1 mL of the urine sample. The enzyme may beβ-glucuronidase or a mixture of β-glucuronidase and arylsulfatase, andthe amount may be in the range of 80 to 140 unit/mL.

The entrapped β-cyclodextrin polymers are added to the sample, and themixture is stirred and centrifuged at 1,500 rpm or higher.

The entrapped β-cyclodextrin polymers are isolated by filtration, and aphosphate buffer solution and at least one polar organic solventselected from the group consisting of tetrahydrofuran (THF), ethylacetate and ether or a mixture are added. The pH of the mixture isadjusted to 9.0 to 10.0 using 5% K₂CO₃ aqueous solution. The adjusted pHrange should be in optimum state in order to extract steroid hormonesfrom the sample upon considering pKa values of an analyte. When the pHis not within the range above, extraction efficiency is considerablydecreased.

The steroid compound is then extracted by shaking the mixture. Here, theorganic solvent may be at least one selected from the group consistingof ethyl acetate, n-hexane and ether or a mixture thereof. The amount ofthe organic solvent is minimized to the extent to separate compoundsdissolved in the polar organic solvent such as THF from the aqueoussolution.

According to the method of extracting steroid compounds of the presentinvention, various compounds having a steroid structure can be easilyextracted, and more particularly, cholesterol, steroid hormonesincluding an endogenous androgen and estrogen, synthetic anabolicsteroids taken by athletes for the purpose of strengthening muscles,corticoids, and the like can be selectively extracted.

The endogenous androgen may be dihydrotestosterone (DHT),dehydroepiandrosterone (DHEA), testosterone, 5α-androstane-3α, 17β-diol,androstenedione, epitestosterone, 5α-androstane-3β, 17β-diol,androstenediol, androsterone, etiocholanolone, 11-keto-androsterone(11-keto-A), 11-keto-etiocholanolone (11-keto-E),11-hydroxy-androsterone (11-OH-A), 11-hydroxy-etiocholanolone (11-OH-E)or 5α-androstanedione, but is not limited thereto.

In addition, the estrogen may be estrone, 17β-estradiol, estriol,2-hydroxy-estrone (2-OH-E1), 2-hydroxy-estradiol (2-OH-E2),17-epiestriol, 4-hydroxy-estrone (4-OH-E1), 4-hydroxy-estradiol(4-OH-E2), 2-methoxy-estrone (2-MeO-E1), 2-methoxy-estradiol (2-MeO-E2)or 16α-hydroxy-estrone (16α-OH-E1), but is not limited thereto.

The synthetic anabolic steroids may be calusterone, bolasterone,boldenone, clostebol, mibolerone, fluoxymesterone, ethisterone,drostanolone, formebolone, 16β-hydroxy-furazabol, methyltestosterone,boldione, α-trenbolone, dianabol, stenbolone, metenolone,19-norandrosterone (19-NA), 19-noretiocholanolone (19-NE), oxandrolone,6β-hydroxy-furinabol, oxymesterone, 3′-hydroxy-stanozolol, norbolethone,ethylestrenol, gestrinone, tetrahydrogestrinone, or metabolites thereof,but is not limited thereto.

In addition, corticoids included in a small amount in urine can also besimultaneously extracted from urine. The corticoids may betriamcinolone, prednisolone, prednisone, fluochlorocortisone,6α-methylprednisolone, betamethasone, dexamethasone, flumethasone,beclomethasone, triamcinolone acetonide, desonide, flunisolide,flurandrenolide, fluocinolone acetonide, desoximethasone, budesonide,flucinonide, amcinonide, cortisol, or cortisone, but is not limitedthereto.

According to the present invention, steroid compounds can be simplyseparated from an inclusion complex of β-cyclodextrin with steroidcompounds using a simple method of forming the complex of β-cyclodextrinand steroid compounds by adding entrapped β-cyclodextrin polymersprepared according to the present invention to the biological samplescontaining various steroid compounds and extracting the steroidcompounds from the complex using an organic solvent.

In addition, the present invention can be efficiently applied toclinical research on various endocrine-dependent diseases since steroidhormones including androgen, estrogen, corticoids, or the like can beselectively extracted from biological fluids such as urine. A sample inwhich the steroid compound is removed can also be prepared.

In addition, doping control of athletes can be efficiently performedaccording to the present invention since synthetic anabolic steroids canbe efficiently extracted.

In addition, the entrapped β-cyclodextrin polymers used to extract thesteroid compounds can be recovered and reused after being washed with anorganic solvent.

The present invention will now be described in more detail withreference to the following examples. The following examples are forillustrative purposes only and are not intended to limit the scope ofthe invention.

Example 1 Method of Preparing Entrapped β-cyclodextrin Polymers

2.5 g of β-cyclodextrin was dissolved in a NaOH solution (2.5 g/7.5 mL),and 4.4 mL of epichlorohydrin was gradually added to the solution for 20minutes. The mixture was stirred at room temperature for 4 hours. Theresultant polymer in a gel state was immersed in a 0.3 M CaCl₂ solutionto be cured. Then, the cured polymer was sufficiently washed with waterand ethanol, dried at 70° C., and pulverized using a pulverizer toprepare entrapped β-cyclodextrin polymers having an average particlediameter of 200 μm (FIG. 2).

Experimental Example 1 Identification of Polymerized β-cyclodextrin

Structures of β-cyclodextrin and entrapped β-cyclodextrin polymersprepared according to Example 1 were identified by 3000× magnificationat 15 kV using a scanning electron microscope (SEM), and the results areshown in FIGS. 3A and 3B.

While the non-polymerized β-cyclodextrin particles were uniformlydistributed as shown in FIG. 3A, the entrapped β-cyclodextrin polymersprepared according to Example 1 were cross-linked to each other as shownin FIG. 3B. In addition, as shown in FIG. 3C, Ca ion particles derivedfrom CaCl₂ were distributed on the surface of β-cyclodextrin polymers.

Example 2 Method of Extracting Steroid Compounds Using Entrappedβ-cyclodextrin Polymers

2 mL of urine was mixed with 1 mL of 0.2 M phosphate buffer solution (pH7.2), and 50 μL of β-glucuronidase was added thereto to hydrolyze theurine. 0.5 g of the entrapped β-cyclodextrin polymers prepared inExample 1 was added thereto, and the mixture was stirred for 10 minutes.Then, the mixture was centrifuged at 3000 rpm for 8 minutes to isolateentrapped β-cyclodextrin polymers.

1 mL of a phosphate buffer solution and 3 mL of tetrahydrofuran (THF)was added to isolated polymers, and the mixture was stirred for 10minutes. 0.7 mL of 0.5% K₂CO₃ solution was added thereto to adjust thepH of the solution to 9.6, and 2 mL of a mixed solution of ethyl acetateand n-hexane (2:3, v/v) was added to the mixture to extract androgen andestrogen contained in the powders.

The extracted androgen and estrogen were reacted with 50 μL of aderivative reagent (MSTFA/NH₄I/DTE, 1000:4:5, v/w/w) at 60° C. for 20minutes, and analyzed by a selected-ion monitoring (SIM) mode usingGC-MS under conditions described below.

-   -   Instrument(s): Agilent 6890 GC and Agilent 5973N Mass Selective        Detector    -   Column: Ultra-2 fused-silica capillary column        -   (Agilent Technologies; length 25 m, inner diameter: 0.2 mm,            thickness: 0.33 μm)    -   Injector Temperature: 280° C.    -   Carrier Gas: Helium (0.8 mL/min)    -   Injection amount: 2 μL    -   Injection Mode: 10:1 split mode    -   Oven Temperature: The temperature of the oven was increased from        200° C. to 260° C. at a rate of 2° C. per minute, maintained for        5 minutes, and increased to 280° C. at a rate of 4° C. per        minute. Then, the temperature was increased to 310° C. at a rate        of 15° C. per minute, and maintained for 3 minutes.    -   Temperatures of Source and Analyzer: 230° C. and 300° C.    -   Ionization: Electron impact ionization (EI)    -   Ionization Energy: 70 eV    -   Characteristic Ions for Analysis: m/z 434, 432, 436, 430, 421,        419, 520, 522, 458, 414, 416, 504, 502, 487, 444, 446

Qualitative chemical analyses of the compounds were performed based on asingle characteristic ion with retention time in the column. 4 to 11ions from each of 6 groups were simultaneously analyzed. The results areshown in FIGS. 4A and 4B.

That is, cholesterol and 15 endogenous androgens, such asdihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), testosterone,5α-androstane-3α, 17β-diol, androstenedione, epitestosterone,5α-androstane-3α,17β-diol, androstenediol, androsterone,etiocholanolone, 11-keto-androsterone (11-keto-A),11-keto-etiocholanolone (11-keto-E), 11-hydroxy-androsterone (11-OH-A),11-hydroxy-etiocholanolone (11-OH-E), and 5α-androstanedione; and 11estrogens, such as estrone, 17β-estradiol, estriol, 2-hydroxy-estrone(2-OH-E1), 2-hydroxy-estradiol (2-OH-E2), 17-epiestriol,4-hydroxy-estrone (4-OH-E1), 4-hydroxy-estradiol (4-OH-E2),2-methoxy-estrone (2-MeO-E1), 2-methoxy-estradiol (2-MeO-E2), and16α-hydroxy-estrone (16α-OH-E1), were selectively extracted from theurine sample. As a result, recovery rates of 15 androgens and 11estrogens were in the range of 92 to 126%.

The recovery rates (same as recovery rates measured in Examples 3 to 4)were calculated by comparing the results of analyses and concentrationsof the same steroid hormones before and after extraction.

It was confirmed that the method of extracting steroid compounds usingentrapped β-cyclodextrin polymers has excellent efficiency as comparedto those of conventional methods disclosed in Journal of ChromatographyA, 885: 3-16, 237-250, 321-341, 2000; and Rapid Communications in MassSpectrometry, 16: 2221-2228, 2002.

In addition, extraction rate of the cholesterol was 96%. In particular,although various technologies of removing cholesterol from samples havebeen reported (Archives of Pharmaceutical Research, 27: 873-877, 2004;and Archives of Pharmaceutical Research, 27: 1183-1187, 2004), themethod of extracting cholesterol using entrapped β-cyclodextrin polymersprepared according to the present invention is known most effective inremoving cholesterol from biological samples with a cholesterol removalrate of up to 98%.

Example 3 Method of Extracting Synthetic Anabolic Steroids UsingEntrapped β-cyclodextrin Polymers

2 mL of urine was mixed with 1 mL of 0.2 M phosphate buffer solution (pH7.2), and 50 μL of β-glucuronidase was added thereto to hydrolyze theurine. 0.5 g of entrapped β-cyclodextrin polymers was added thereto, andthe mixture was stirred for 10 minutes. Then, the mixture wascentrifuged at 3000 rpm for 8 minutes to isolate polymers. 1 mL of aphosphate buffer solution and 3 mL of THF were added to the separatedpowders, and the mixture was stirred for 10 minutes. Then, 0.7 mL of0.5% K₂CO₃ solution was added thereto to adjust the pH of the solutionto 9.6, and 2 mL of diethylether was added to the mixture to extractsynthetic anabolic steroids contained in the powders. The extractedsynthetic anabolic steroids were reacted with 50 μL of a polymerizationreagent (MSTFA/NH₄I/DTE, 1000:4:5, v/w/w) at 60° C. for 20 minutes, andanalyzed using GC-SIM/MS under conditions described below.

-   -   Instrument(s): Agilent 6890 GC and Agilent 5975 Mass Selective        Detector    -   Column: Ultra-1 fused-silica capillary column        -   (Agilent Technologies; length 17 m, inner diameter 0.2 mm,            thickness 0.11 μm)    -   Injector Temperature: 280° C.    -   Carrier Gas: Helium (0.6 mL/min)    -   Injection Amount: 2 μL    -   Injection Mode: 10:1 split mode    -   Oven Temperature: The temperature of the oven was increased from        180° C. to 260° C. at a rate of 4° C. per minute, increased to        320° C. at a rate of 15° C. per minute, and maintained for 3.67        minutes.    -   Temperatures of Source and Analyzer: 230° C. and 300° C.    -   Ionization: Electron impact ionization (EI)    -   Ionization Energy: 70 eV    -   Characteristic Ions for Analysis: Two characteristic ions for a        compound which is capable of representing their chemical        structures were selected, and 11 groups were analyzed under        conditions described below. 1 (14), 2 (21), 3 (21), 4 (20), 5        (20), 6 (25), 7 (21), 8 (30), 9 (27), 10 (23), 11 (25)

Qualitative chemical analyses of the compounds were performed based ontwo characteristic ions with retention time in the column. The resultsare shown in FIGS. 5A, 5B, 5C and 5D.

That is, 30 synthetic anabolic steroids such as calusterone,bolasterone, boldenone, clostebol, mibolerone, fluoxymesterone,ethisterone, drostanolone, formebolone, 16β-hydroxy-furazabol,methyltestosterone, boldione, α-trenbolone, dianabol, stenbolone,metenolone, 19-norandrosterone (19-NA), 19-noretiocholanolone (19-NE),oxandrolone, 6β-hydroxy-furinabol, oxymesterone, 3′-hydroxy-stanozolol,norbolethone, ethylestrenol, or metabolites thereof were selectivelyextracted from the urine sample for doping tests of athletes. As aresult, extraction rates of the synthetic anabolic steroids were in therange of 84 to 131%.

The extraction rates of the synthetic anabolic steroids included inurine which was obtained in Example 3 had constant values compared toextraction rates obtained according to conventional SPE in which theextraction rates vary according to polarity of compounds

Particularly, extraction rates of steroid hormones having high polaritysuch as metabolites of fluoxymesterone and oxandrolone were about 40% inSPE using hydrophobic interaction, while extraction rates thereof werein the range of 91 to 105% according to Example 3. Thus, it wasconfirmed a high yield could be obtained not by polarity of compoundsbut structural properties of compounds.

Example 4 Method of Extracting Synthetic Anabolic Steroids andCorticoids Using Entrapped β-cyclodextrin Polymers

2 mL of urine was mixed with 1 mL of 0.2 M phosphate buffer solution (pH7.2), and 50 μL of β-glucuronidase was added thereto to hydrolyze theurine. 0.5 g of entrapped β-cyclodextrin polymers was added thereto, andthe mixture was stirred for 10 minutes. Then, the mixture wascentrifuged at 3000 rpm for 8 minutes to isolate the polymers.

1 mL of a phosphate buffer solution and 3 mL of THF were added to theseparated entrapped β-cyclodextrin polymers, and the mixture was stirredfor 10 minutes. Then, 0.7 mL of 0.5% K₂CO₃ solution was added thereto toadjust the pH of the solution to 9.6, and 2 mL of diethylether was addedto the mixture to extract corticoids contained in the entrappedβ-cyclodextrin polymers. The extracted corticoids were dissolved in 100μL of 50% acetonitrile solution prepared by 0.1% of acetic acid, andanalyzed using LC-tandem mass spectrometry (LC-MS/MS) under conditionsdescribed below.

-   -   Instrument(s): Surveyor HPLC & TSQ Quantum Discovery MAX    -   Column: Hypersil Gold    -   (ThermoFinnigan; length 50 mm, inner diameter: 2.1 mm, particle        size: 1.9 μm)    -   Mobile Phase: A (0.1% acetic acid in 5% acetonitrile)        -   B (0.06% acetic acid in 95% acetonitrile)    -   Injection Amount: 5 μL    -   Ionization: electrospray ionization (ESI) in the positive mode    -   Analysis Mode: Single Reaction Monitoring (SRM), 0.02 sec/scan    -   Spray Voltage: 4500 V    -   Peak Width: Q1 (0.5) & Q3 (0.7)    -   Velocity of Mobile Phase: 0.3 mL/min    -   Mobile Phase Change: Mobile phase was changed from initial state        of 90% (A) to 10% (A) for 2 minutes, maintained for 1 minute,        and returned to the initial state of 90% (A) for 5 minutes. The        analysis results of two synthetic anabolic steroids (17:        gestrinone and 20: tetrahydrogestrinone) and 21 corticoids        included in the urine sample were shown in FIG. 6.

That is, extraction rates of two synthetic anabolic steroids (17:gestrinone and 20: tetrahydrogestrinone) and 21 corticoids (1:triamcinolone, 2: prednisolone, 3: prednisone, 4: fluochlorocortisone,5: 6α-methyly-prednisolone, 7: betamethasone & dexamethasone, 8:flumethasone, 9: beclomethasone, 10: triamcinolone acetonide, 11:desonide, 12: flunisolide, 13: flurandrenolide, 14: fluocinoloneacetonide, 15: desoximethasone, 16: budesonide, 18: flucinonide, 19:amcinonide, 21: cortisol, 22: cortisone, 12: internal standard, andd₄-cortisol) included in the urine sample were in the range of 72 to119%. It was confirmed that the results were complimentary when comparedto a conventional liquid-liquid extraction (Rapid Communications in MassSpectrometry, 17: 2107-2114, 2003).

Example 5 Packing Material for Solid-Phase Extraction Using CuredPolymerized β-cyclodextrin Powders

Solid-phase extraction was performed using the cured polymerizedβ-cyclodextrin powders having an average diameter of 200 μm prepared inExample 1 as a packing material. As a result, swelling did not occur.

Comparative Example Packing Material for Solid-Phase Extraction UsingEntrapped β-cyclodextrin Polymers

Solid-phase extraction was performed using entrapped β-cyclodextrinpolymers with an average diameter of 200 μm which was not entrapped withmetal ions as a packing material. As a result, swelling occurred.

As describe above, according to the present invention, various types ofsteroid compounds can be selectively separated from the samples forclinical trials, food analyses, environmental analyses, drug tests, anddoping control of athletes by a simple process.

In addition, entrapped β-cyclodextrin polymers prepared according to thepresent invention can be used as a packing material for solid-phaseextraction. When steroid compounds are extracted, steroid compounds canbe simply extracted by adding an organic solvent to a complex ofβ-cyclodextrin and steroid compounds without using an additional device,which is distinct from conventional solid-phase extraction.

In addition, the entrapped β-cyclodextrin polymers have high economicalvalues since the powders can be washed with an organic solvent andreused.

While the present invention has been particularly shown and describedwith reference to exemplary embodiments thereof, it will be understoodby those of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the present invention as defined by the following claims.

1. Entrapped β-cyclodextrin polymers prepared by the reaction of β-cyclodextrin monomers and epichlorohydrin followed by entrapment with metal ions.
 2. The entrapped β-cyclodextrin polymers of claim 1, wherein the metal ions are Ca or Na.
 3. A method of preparing entrapped β-cyclodextrin polymers, the method comprising: preparing β-cyclodextrin polymers in a gel state by dissolving β-cyclodextrin in an aqueous alkali solution and adding epichlorohydrin to the solution; preparing entrapped β-cyclodextrin polymers by immersing the β-cyclodextrin polymers in a gel state in a metal salt solution followed by entrapping on the surface of the β-cyclodextrin polymers; and washing, drying and pulverizing the entrapped polymers to powderize the entrapped β-cyclodextrin polymers.
 4. The method of claim 3, wherein the alkali aqueous solution is prepared using an alkali metal hydroxide.
 5. The method of claim 4, wherein the alkali metal hydroxide is NaOH or KOH.
 6. The method of claim 3, wherein the metal salt is selected from the group consisting of CaCl₂, NaCl, Ca(NO₃)₂ and CaSO₄.
 7. The method of claim 3, wherein the concentration of the metal salt solution is in the range of 0.2 to 0.3 M.
 8. A packing material for solid-phase extraction comprising entrapped β-cyclodextrin polymers according to claim
 1. 9. A packing material for solid-phase extraction comprising entrapped β-cyclodextrin polymers according to claim
 2. 